This proposal is submitted to pursue the exploration of the pathogenesis of Parkinson's disease (PD). Pertinent to this goal, we have found that cyclooxygenase-2 (COX-2), the rate-limiting enzyme in prostaglandin E2 (PGE2) synthesis, is upregulated in nigrostriatal dopamine (DA)-containing neurons in post-mortem PD tissues and in the 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP) mouse model of PD. We have also shown that both ablation and inhibition of COX-2 protect against MPTP neurotoxicity via a non-inflammatory process. Given this, we now wish to examine first whether the deleterious effects of COX-2 depend upon cytosolic phospholipase A2 (cPLA2), as this enzyme produces COX-2's main substrate. Specific Aim (SA)-I will thus define the temporal and anatomical relationship between cPLA2 and COX-2 in MPTP mice and in PD tissues. A comparison of MPTP neurotoxicity between cPLA2 deficient mice treated with and without a COX-2 inhibitor will also be done. Second, it is also known that DA stimulates PGE2 production, which suggests that this neurotransmitter could exacerbate COX-2's effects on nigrostdatal DA neurons. SA-II will thus assess the effects of DA on cPLA2 and COX-2-activities and on neuronal death in primary ventral midbrain cultures exposed to MPTP's toxic metabolite. In addition, MPTP neurotoxicity will be compared between wild-type and knockout COX-2 mice pre-treated with drugs that increase or decrease brain DA. Third, PGE2 produced by injured nigrostriatal DA neurons may activate PGE2-EP3 receptors that are present on many neurons. To ascertain whether such a mechanism is operative here, SA-III will define EP3 receptor distribution in MPTP mice and in PD tissues; because EP3 receptors can be expressed on both plasma and nuclear membranes, EP3 subcellular distribution in MPTP mice will also be studied. Since activation of nuclear EP3 alters intranuclear calcium uptake and c-los transcription, occurrence of these two events will be determined in nigrostriatal DA neurons after MPTP injection. Evaluation of MPTP neurotoxicity in EP3-deficient mice or in COX-2-deficient mice treated with an EP3 agonist will complete SA-III investigations. Together, these studies should provide a deeper understanding of how COX-2 may contribute to the pathogenesis of PD and to the selective nature of the neurodegenerative process in this disease.